Grades 11 12 Biotech Boot Camp 2015 Kirk

Grades 11 -12 Biotech Boot Camp 2015 Kirk Brown Director of Science & STEM Integration & Innovation S 2 I 2 San Joaquin County Office of Education Andrew Hutton B. S. Neuroscience/ B. A. Psychology University of California, Santa Cruz

Agenda for Day 1 1. 2. 3. 4. 5. 6. 7. 8. Introductions Safety Orientation Lab Notebook Orientation Pipetting Review and Practice Solution Making 1. Percent Solutions 2. Molar Solutions Lunch Ouchterlony Immuno-diffusion Test Detecting GMO in Dorito Chips Part 1

Notebooks u u Look at notebooks Review Rubric Use as a tool to record what you do as you do it Some tips

Pipetting • • • Transfer Pipet Use Goals: Transfer 250μl, 500μl, 750μl and 1 ml of colored water 24 well plate. Each partner will complete task How might you measure the accuracy?

Micropipetting Micopipetting Video • Goals: 1. Pipet a series of droplets on a piece of wax paper. • 2, 4, 6, 8, 10, 12 μl droplets in order 2. 3. 4. Show Instructor Write your initials in 3 μl droplets Show instructor

Micropipetting Goals: 1. Pipet 45 μl of colored water into a 1. 5 ml microcentrifuge tube. 2. Transfer 5 μl into a new 1. 5 ml microcentrifuge tube 3. Transfer 15 μl of colored water from the 1. 5 ml tube into the 1. 5 ml tube that already has 5 μl in it. •

Making Solutions • Percent Solutions

Making Solutions • Molar Solutions

GMO Detection by PCR • Research • question: Does the food tested contain genetically modified plant material? • Objectives: • • • Grind one test food and one non-GMO control food using a mortar and pestle Extract g. DNA with Insta. Gene matrix Set up six PCR reactions with test food g. DNA, g. DNA from non-GM control food and GM-positive plasmid DNA using GMO master mix (red) and plant master mix (green) for each sample Load and run PCR products on 3% agarose TAE gel Determine whether the test food has been genetically modified

Safety Reminders • Follow all laboratory safety procedures Wear appropriate PPE • Be careful when using 10% bleach, it can harm clothes and is hazardous • Remember to balance centrifuge and ensure inner lid is on • Be careful with electricity in the presence of liquids • If ethidium bromide or SYBR Safe stain are used, follow appropriate safety precautions •

Skills to Master • Use mortar and pestle • Perform DNA extractions using Insta. Gene matrix • Set up PCR reactions • Use a thermal cycler • Perform agarose gel electrophoresis • Analyze results on an agarose gel

• Tips • • PCR is very sensitive; watch for contamination of reagents or tips Mortar and pestle is a source of contamination and should be wiped with 10% bleach and rinsed with tap water followed by distilled water Too much plant material can overwhelm the Insta. Gene matrix. Use only the recommended amount Use a transfer pipet to transfer the ground food to the Insta. Gene matrix since micropipet tips are easily clogged Use aerosol barrier tips when setting up PCR reactions Keep master mix and reaction tubes on ice until they are ready to be placed in thermal cycler Mix reagents thoroughly and ensure reactions are at bottom of tubes

Student Workstation Materials

Student Workstation Materials

Protocol Highlights/Tips • • Use the proper amount of plant material and dilute with 5 ml of water for every gram of plant material Use a transfer pipet to transfer plant material to Insta. Gene matrix Use aerosol barrier pipet tips Keep tubes on ice Keep a map of tube location in thermal cycler Ensure that reagents are mixed well and on bottom of tubes Minimize contamination 3% TAE gels should be used to run samples

Using PCR to Detect GM Sequences • Play video: GMO Detection by PCR

• Gel Results • • Compare bands to the PCR MW standard Compare lanes 1, 3 and 5 to see if plant DNA was amplified • • Since lane 1 and 5 are controls, both should amplify plant DNA Compare lanes 2, 4 and 6 • Since lane 6 is a positive control, and lane 2 is a negative control, compare the results of lane 4 to both

• Summary • Make sure to: • • • Record all steps in your notebook Keep tubes on ice and watch for contamination Record the location of samples in thermal cycler Run samples in a 3% gel due to their small length Compare PCR products to determine if the plant material was genetically modified or not

Ouchterlony Double Immunodiffusion Assay • Research • question: Which of the test samples is from chicken? • Objectives: • • • Prepare the Ouchterlony assay plate Add the antibody and the test samples to the plate Interpret the results

Safety Reminders • Follow all laboratory safety procedures Wear appropriate PPE • Take proper precautions when handling raw meat juices, especially chicken • Disinfect all surfaces that come into contact with raw meat or blood proteins • Wash hands thoroughly after any contact with raw meat or blood proteins • Read and become familiar with all MSDSs for this activity •

Skills to Master § Perform an Ouchterlony double immunodiffusion assay

Tips • Be sure to change tips between samples and reagents to avoid sample cross contamination • When making the wells, it is helpful to depress the bulb on the cut transfer pipet before inserting into the PBS agarose • Release the bulb slightly and then wait until the plug comes into the transfer pipet • Plates can be incubated in a small tub with wet paper towels agar side down until all of the sample has absorbed into the agarose

Student Workstation Materials

Determining Antibody and Antigen Interaction • Play video: Ouchterlony Assay

Protocol Highlights/Tips • • Cut a transfer pipet on a cutting board so that the pipet sizes are parallel and no graduations are in the way Punch the holes 1 cm from the center whole as shown in protocol Change tips between samples to avoid contamination It may take two days to clearly show precipitation lines

Results • Precipitation the wells lines can be seen between

Summary • Make sure to: Record all steps in your notebook • Make sure the wells are 1 cm from the center and clearly labeled on the plate • Place the plates into a tub with a lid that has moist paper towels in it to present drying • It may take up to two days for the precipitation line to show up •

Today’s + and Δ • What did you learn? • What did you like about today? • What would you like to change or modify? • Homework! • Only if you want to!

Agenda for Day 2 1. 2. 3. 4. 5. 6. GMO PCR Gels Gel Analysis PCR Background Lunch PV-92 Chelex Extraction/PCR Set-up Bacterial Plate counts and serial dilution

Loading a Gel • Add 4 μl of 6 x Uview Loading Dye to each sample. • Mix by pipetting • Load 20 μl in each well Lane Contents 1 Neg Control: PMM 2 Neg Control: GMM 3 Test: PMM 4 Test: GMM 5 Pos Control: PMM 6 Pos Control: GMM 7 PCR MW Standard

Agarose Electrophoresis • • Power Supply Electrical current carries negativelycharged DNA through gel towards positive (red) electrode Loading a gel Buffer Dyes Agarose gel

• Pouring an Agarose Gel • Casting a Gel

• Deoxyribonucleic Acid (DNA)

DNA Diagram

Human Modeling of Nucleotide Phosphate Base Sugar

How PCR Works • http: //www. bio- rad. com/webroot/web/movies/lse/product s/programs/04 -0522_PV 92_PCR. html

Detection of the Human PV 92 Alu Insertion • Research • • questions: What is the genotype for the PV 92 Alu insertion on chromosome 16 of the samples in your team? What is the frequency of the PV 92 Alu element in the class? • Objectives: • • • Collect a cheek cell or hair follicle sample Extract g. DNA with Insta. Gene matrix Set up a PCR reaction Load and run PCR products on a 1% agarose TAE gel Determine the genotype of DNA samples Calculate the frequency of PV 92 Alu element in the class population

Safety Reminders • Follow all laboratory safety procedures Wear appropriate PPE • Handle only your own saliva sample and not others • Remember to balance centrifuge and ensure inner lid is on • Be careful with electricity in the presence of liquids • If ethidium bromide or SYBR Safe stain are used, follow appropriate safety precautions •

Skills to Master • Perform DNA extractions using Insta. Gene matrix. • Set up PCR reactions Use a thermal cycler • Perform agarose gel electrophoresis Analyze an agarose gel

Tips • • When extracting cheek cells, gently chew the inside of mouth to help loosen epithelial cells while swishing with saline A small pellet of cells should be visible once the samples have been centrifuged. If not, add more mouth rinse and repeat centrifugation When samples are incubated at 56ºC, shake them after 5 minutes. This ensures that more cells break open at 95ºC PCR is very sensitive; watch for contamination of reagents or tips Use aerosol barrier tips when setting up PCR reactions Keep master mix and reaction tubes on ice until they are ready to be placed in thermal cycler Mix reagents thoroughly and ensure reactions are at bottom of tubes

Student Workstation Materials

Student Workstation Materials

Determining Alu PV 92 Genotype by PCR • Play video: Alu PV 92 Detection by PCR

Protocol Highlights/Tips • • Chew cheeks for maximum extraction Be careful not to pour out pellet Use aerosol barrier pipet tips Keep tubes on ice Keep a map of tube location in thermal cycler Ensure that reagents are mixed well and on bottom of tubes Minimize contamination 1% TAE gels should be used to run samples

Estimating Bacterial Numbers Serial Dilutions • Direct Measurements of Bacterial numbers • Plate counts: Perform serial dilutions of a sample

Serial Dilution 10 μl 90 μl of broth in each tube 10 μl

Plating the Serial Dilutions 100 μl 100 μl

Determining Bacterial Concentration • Play video: Serial Dilution and Plate Counts

Today’s + and Δ • What did you learn? • Add two more words to the word wall… • What did you like about today? • What would you like to change or modify?

Agenda for Day 3 1. 2. 3. 4. 5. 6. 7. PV-92 Gels Gel Analysis Bacterial Quantitation Results Lunch Gram Staining Clean-up Graduation and Certificates

Student Workstation Materials

Gel Results • • Compare bands in lanes 5– 8 to three controls Always ensure that the bands match the controls and use the EZ load mass ruler as a secondary control to ensure they are approximately 960 bp for (+) and 660 bp (-)

Calculate the number of CFU/ml Concentration of Bacteria (CFU/ml) = • Find a plate that has between 20 and 200 colonies • If the 1/1, 000 plate has 32 colonies and 100 µl was plated then the following calculation would determine the CFU/ml • First step is to multiply the CFU by dilution factor: 32 CFU x 1, 000 Answer in CFU/ml x = Amount plated Convert µl to ml

Gram Staining • Research questions: • • • Are bacteria found in yogurt and E. Coli HB 101 bacteria gram-positive or gram-negative? What are the size and shape of E. coli HB 101 and bacteria from yogurt? Objectives: • • • Mount bacteria on a microscope slide using aseptic technique Perform Gram staining of bacteria Determine gram status of bacteria Determine cell shape of bacteria Determine size of bacteria

Safety Reminders • Follow all laboratory safety procedures Review all MSDSs of the stains used in the activity • Gram staining has flammable components; ensure no open flames or spark hazards are present when Gram staining • Use aseptic technique when using bacteria • Wear appropriate PPE • Do not eat or drink in the laboratory •

Skills to Master • Perform Gram staining of bacterial • Heat fix bacteria to slide • Observe bacteria using a microscope • Differentiate gram-positive and gramnegative bacteria • Identify bacteria from cell shape • Sketch microscopic details • Estimate size of cells using a microscope

Tips • Wear gloves to avoid staining fingers • Have multiple beakers of water to use for destaining • Wooden clothes pins can be used to hold slides to heat fix and stain • The decolorizer used in Gram staining has a high percentage of alcohol and no flames should be present when using • Wear appropriate PPE

Student Workstation Materials

Gram Staining Bacteria • Play video: Gram Staining

Protocol Highlights/Tips • • • Remember to use appropriate safety equipment Make sure the bacteria are dry on the slide before heat fixing Follow aseptic technique at all times Make sure that slide is completely dry after Gram staining before observing in the microscope Make detailed observations in your notebook Digital cameras and cell phones can be used to take pictures of bacteria in microscope through the eyepiece

Summary • Make sure to: • • Record all steps in your notebook Make detailed observations after each step If oil immersion lenses are available, use to view Gram stained bacteria at 1, 000 x Bleach or autoclave plates/tubes upon the completion of the experiment

Clean Up!

Thanks and Graduation! • Amy Kennedy • Ann Carl • Bret States • Jane Steinkamp • James Mousalimas • You and your parents • Next Steps: • Next Summer…. • Helper